Method of polymyxin b recovery from fermentation broth

ABSTRACT

The invention is related to the method of polymyxine B recovery from fermentation broth for the purpose of pure substance recovery. The invention mentioned above is obtained by using the method of polymyxine B recovery from fermentation broth according invention, which abstract is characterized by, that the filtrate is obtained from fermentation broth, its pH and color is adjusted and purified on non-ionogenic synthetic adsorbents of polystyrene type with specific surface 500 till 1000 m 2 .g −1  and fabric size 3 till 30 nm. The polymyxine B is eluated from adsorbent by aqueous solution of organic solvent. The polymyxine B base is coagulated from eluate after adjustment of pH to alcaline area. The polymyxine B base is converted by mineral acid to polymyxine B salt solution, from which is obtained crystal substance by drying.

TECHNICAL FIELD

The invention is related to method of polymyxine B recovery fromfermentation broth.

BACKGROUND ART

The polymyxine is a complex of very similar polypeptide antibioticsisolated from different types of Bacillus polymyxa strains and relativespecies. It is cyclic polypeptide with free aminoacides groupscontaining characteristic constituents such as α,γ-aminobutanic acid,L-treonine and fatty acides (6-methyloktanoyl acid,3-hydroxy-6-metyloktanoyl acid, 6-metylheptanoyl acid, heptanoyl acid,oktanoyl acid, nonanoyl acid). This complex was divided bysemi-preparative PLRP-S with reverse faze on 7 components (Orwa J. A.,Govaert C., Busson R., et all, 2001, J.Chromatography A, 912, 369-373).The structure was characterized by methods ¹H- and ¹³C -NMR andmolecular weight was measured by mass spectroscopy method. It wasconfirmed structure of polymyxine B₁, Ile-B₁, B₂, B₃ and B₄. The furthertwo components, polymyxine B₅ and B₆, was isolated for the first timeand characterized at this study.

The polymyxine B and E have lower toxicity like another polymyxine andthey are preferred for medical use, mainly antibacterial more effectivepolymyxine B.

The procedures for recovery of polymyxine from fermentation broth inprincipal are based on application of activated carbon and ionsexchange. In U.S. Pat. No. 2,565,057, is granted and described acharacter of polypeptide antibiotic base as a metabolic products ofBacillus aerosporus and Bacillus polymyxa, there are listed also methodsof his recovery from cultivation media by using adsorption character ofactivated carbon. The first part of impurities are separate from thefiltrate of the broth by activated carbon in acid area (pH 2.5). Thediscolored solution of antibiotic is adjust to neutral area and than itis adsorbed on active carbon. The adsorbed antibiotic is eluated byaqueous solution of acid acetone. The eluate is purified by adjustmentof pH, separation of acetone and re-adsorption on activated carbon andneutralization of eluate with CaCO₃ and finalization by lyofilisation ofantibiotic solution. Another alternative of antibiotic purification fromeluate is preparation of insoluble salts such as coagulation by picricacid and reconvert of this salt into aqueous soluble salts usually in aform of sulphates (M. Harold et all.: Antibiotika {hacek over (C)}sAVPraha, 1957, page 285-288).

In further 2 patents (GB 742 589 and GB 782 926) the filtrates from thefermentation broth are purified in weak acid ions exchangers indifferent column systems. The adsorbed polymyxine is eluated by solutionof mineral acid or buffer solution with pH in a range from 3 to 5. Theobtained eluate is purified on anion exchanger or in strong acid cationexchanger. This repurify eluate is concentrated and polymyxine iscoagulated by addition of alcaline solution. Coagulation is running in arange of temperature from 60° C. to 90° C.

U.S. Pat. No. 3,132,994 (1964) describe the method of increasepurification crude sulphates—and N-methylsulphonates of polymyxine B andE. The background of this method is oxidation of this salts solutions inacid or neutral pH (pH 3.5 till 7.5) with 1% water solution of KMnO₄. Inthe next step the low molecular weight fabrics are separate, includingMn-cations in a strong acid cation exchanger in H⁺ cycle, where thepolymyxine is not adsorbed.

The procedure for recovery and purification of amphotem and alcalineantibiotics including polymyxine B, using reactive extraction intoorganic solvent limited with aqueous mixture and extraction in a presentof holders di-(2-ethylhexyl)-ester or dinonyl ester of phosphoric acidis describe in patent GB 979 887 (1964). Polymyxine B is extracted fromthe broth into 1% solution of di-(2-ethylhexyl)-ester of phosphoric acidin buthylacetate at pH 7. Purification of polymyxines B and E from acidsolutions by alcaline reagent in a present of chelating agents to formcomplexes of cations Ca, Mg, Mn, Fe is describe in patents GB 1089765(1964) and U.S. Pat. No. 3,413,398. The antibiotic are precipitate in aform of base solution by using alcaline agents such as hydroxides andcarbonates of alcaline metals and ammonium hydroxide at pH 8.5 till 11and addition of chelating agents avoid coprecipitation of impurities.There is described the whole list of chelating agents, which used to beadded in equivalent ratio to present polyvalent inorganic ions. Theprecipitation of antibiotic base is able to do in 10% water solution ofacetone. In the examples of these patents are describe method forpurification of polymyxine B, where the acid solution of crudepolymyxine B is under oxidation with KMnO₄, according to the patent GB991602. The leftover of KMnO₄ is eliminated by H₂O₂. The further iscontinued in precipitation of base in present of ethylenediamine-N, N,N, N′ tetraacetic acid tetra sodium salt. Base is filtered off, washedand vacuum-dried. The base is converted to the sulphate of polymyxine Bby molar equivalent of H₂SO₄.

In describe procedures of recovery of polymyxine B solid substance fromthe broth, the broth is purified by complicated methods and crudeantibiotic is obtained. The crude antibiotic must be purified in severalsteps to get pure substance.

DISCLOSURE OF THE INVENTION

Listed disadvantages eliminates the method of polymyxine B recovery fromfermentation broth according to the invention, abstract of its is, thatthe filtrate of the fermentation broth is purificated on non-ionogenicsynthetic adsorbents of polystyrene types, polymyxine B is eluated fromadsorbent by aqueous solution of organic solvent, the base of polymyxineB is precipitated from eluate, than it is converted by mineral acid tomineral salt of polymyxine B, from whom is obtained crystal substance bydrying.

The biomass and insoluble components of cultivation media are separated.The obtained filtrate is adjust to a value pH from 2 to 6 and eventuallydiscolored by activated carbon in amount 1 till 5 g per 1dm³ of filtratebefore next treatment. Discolored or un-discolored filtrate an pH 2 till6, preferable pH 2.5 till 3.5 is purified on non-ionogenic syntheticpolystyrene adsorbents with specific surface 500 till 1000 m².g⁻¹ andfiber size 3 to 30 nm.

The adsorbed polymyxine B is eluated by non-acid or acid aqueoussolution of organic solvent with concentration 20 to 60% at a pH 2 till4, particularly with lower alcohol or ketone, preferable with methanol,ethanol, propanol, secondary propanol and acetone.

>From obtained water-organic eluate, or from eluate after partial ortotal separation of organic solvent, polymyxine B base is coagulated byaddition of water solution of mineral alkaline agent, preferable NaOH,KOH, NH₄OH at a pH 8 till 12, preferable at a pH 9 till 11. The base iscoagulated at a temperature 20 till 60° C. The coagulated polymyxine Bbase is filtered off and washed by deionized water at temperature 30till 80° C. Into washed polymyxine B base, eventually into watersuspension of base, solution of mineral acid is added, such as HCl,H₂SO₄, to a value pH 5 till 7.5. The obtained solution of polymyxine Bis eventually discolored by activated carbon in amount 0.1 till 0.5 gper g of polymyxine B and pure solution of substance is drying.

The invention is supplemented by examples, which did not limit hisproportion.

EXAMPLES OF EMBODIMENTS Example 1

The biomass was separated by centrifugation from 2.1 dm³ of fermentationbroth with 3.1 g of polymyxine B. Supernatant was acidified with 10%solution of H₂SO₄ to pH 3, activated carbon was added in amount 4 g andafter 30 minutes suspension was filtered out. Discolored clear filtratewas percolated through column fulfilled with 500 ml of non-ionogenicadsorbent Diaion HP 21, with flow rate 2 dm³.h⁻¹. The column was washedafter adsorption by 500 ml of deionized water. The adsorbed polymyxine Bwas eluated with 40% aqueous solution of acetone under flow rate 0.5 dm³.h⁻¹. It was obtained 0.84 dm³ of eluate with amount 2.2 g of polymyxineB. Into the eluate was added under very slow agitation at laboratorytemperature 20% aqueous solution of NaOH to a value pH 11. After 2 h ofcrystallization the polymyxine B base was filtered off and washed with 2dm³ of deionized hot water at the temperature 70° C. It was obtained 20g of moist product of polymyxine B base, which was mixed together with 5ml of deionized water and 10% solution of H₂SO₄ was slowly added tillpolymyxine B sulphate occurred. Into 50 ml of polymyxine sulphatesolution at a pH 6 was added 0.5 g of activated carbon and after 45 minsuspension was filtered off. Discolored solution was than filteredthrough the membrane with fiber size 0.45 μm and 0.22 μm. The productwas recovered by freeze-drying. It was obtained 1.7 g of polymyxine Bsulphate with purity 7900 I.U./mg

Example 2

From the eluate which was prepared according to example 1, acetone wasseparated by vacuum distillation. It was obtained 0.5 dm³ ofconcentrate, which was adjust to pH 7.5 with concentrated NH₄OH at thetemperature 60° C. and than was added 20% solution of NaOH till thefinished pH 12. After 2 hours of crystallization at a temperature 40° C.was obtained polymyxine base, which was filtered off and washed 5 timeswith 0.3 dm³ of deionized water at the temperature 30° C. It wasobtained 15 g of moistly coagulate, which was recovered according toexample 1. It was obtained 1.3 g of polymyxine B sulphate with purity8100 I.U./mg.

Example 3

Procedure according to example 1 with following difference that it wasused 60% ethanol for elution. It was obtained 0.58 dm³ of eluate, whichwas recovered according to example 1 with the difference, thatpolymyxine base was precipitated by NH₄OH to a value pH 8. It wasobtained 1.2 g of polymyxine B sulphate with purity 7200 I.U./mg

Example 4

The biomass and insoluble parts were filtered off by vacuum filtrationfrom 2.1 dm³ of fermentation broth with amount of polymyxine B 2.8 g.The filtrate was adjusted by NaOH at a pH 6 and it was adsorbed tocolumn fulfilled with adsorbent Sepabeads SP 207.

After washing of column by deionized water antibiotics was eluated by20% solution of ethanol acidified with H₂SO₄ to a value pH 2.5. Into 1.2dm³ of eluate was added under mixing 20% aqueous solution of KOH to avalue pH 8. It was occurred polymyxine base which was filtered off andwashed 3 times by 0.1 dm³ of deionized water at the temperature 30° C.Into the 12 g of moist polymyxine B base was added under stirring veryslowly 10% solution of H₂SO₄ to the pH 5while the polymyxine B sulphatewas occurred. 40 ml of polymyxine B sulphate solution was discolored by0.7 g of active carbon, filtered off through the 0.22 μm membrane andlyofilizated. It was obtained 1.25 g of polymyxine B sulphate withpurity 7600 I.U./mg

Example 5

From 4 dm³ of fermentation broth with amount 4.4 g of polymyxine B andthe pH 6 was obtained by micro filtration 4.2 dm³ of clear permeate,which was acidified by H₂SO₄ at the pH 3. Into the solution was added 21g of activated carbon and after 1 hour of mixing suspension was filteredoff. Discolored permeate was percolated through the column fulfilled by1.2 dm³ of non-ionogenic adsorbent Amberlite XAD-4. After washing ofcolumn by deionized water the polymyxine B was eluated by 60% watersolution of secondary propanol. It was obtained 1.2 dm³ of eluate, whichwas adjust to a value pH 10.8 by solution of NaOH. The base which wasoccurred was separated by centrifugation and washed by hot deionizedwater while the pH of washed water is 7.8. It was obtained 25 g ofmoisty base which was mixed together with 10 ml of deionized water. Thesolution of 10% H₂SO₄ was slowly added till polymyxine B sulphatesolution was occurred at the pH 7. Solution of polymyxine B sulphate (70ml) was discolored by 0.3 g of active carbon, filtered through themembrane 0.22 μm and lyofilizated. It was obtained 2.4 g of polymyxine Bsulphate with purity 7700 I.U./mg.

Industrial Applicability

The method of polymyxine B recovery according to invention is able touse for recovery of pure antibiotic from fermentation broth.

1. A method of polymyxine B recovery from fermentation broth comprising:(a) obtaining a filtrate from a fermentation broth purified on one ormore non-ionogenic synthetic adsorbents of polystyrene type with aspecific surface of 500 to 1000 m².g⁻¹ and a pore radius of 3 to 30 nm;(b) eluting polymyxine B from the adsorbent with an aqueous solution ofan organic solvent to form an eluate, (c) coagulating the polymyxine Bfrom the eluate; (d) adding mineral acid into to the eluate to formpolymyxine B solution; and (e) drying the polymyxine B solution to forma crystal substance.
 2. The method according to claim 1 wherein thefiltrate, of step (a) is adjusted to a pH of 2 to
 6. 3. The methodaccording to claim 1, wherein the aqueous solution of organic solvent ofstep (b) comprises a lower alcohol or cetone at a concentration of 20 to60% by weight.
 4. The method according to claim 1 further comprisingpartially or completely separating the organic solvent from the eluateof step (b) by adjustng the pH of the eluate to 8 to 12 at a temperatureof 20 to 60° C.
 5. The method according to claim 1 wherein mineral acidis added in step (d) until the pH of solution is from 5 to 7.5.
 6. Themethod of claim 2, wherein the filtrate is discolored using activatedcarbon in amount of 1 to 5 g per 1 dm³ of filtrate.
 7. The method ofclaim 3, wherein the organic solvent comprises methanol, ethanol,propanol, secondary propanol, acetone or combinations thereof.